The long-term goal of this proposal is to understand, in precise molecular terms, how expression of a large set of genes in S. cerevisiae is repressed by Tup1/Ssn6 repressor complex. The proposal is divided into three basic parts: (1) the determination of the mechanism of transcriptional repression by Tup1/Ssn6 and the identification of additional proteins required to carry it out; (2) the development, using Tup1/Ssn6 as the experimental system, of a general model for understanding two common protein motifs, the WD40 repeat and TPR; and (3) a description of the entire Tup1/Ssn6 regulatory network in yeast, including all the DNA-binding proteins that utilize Tup1/Ssn6 and the complete set of genes controlled by each DNA-binding protein. The proposal also covers a new research direction in the lab, an exploration of CHD1, a highly conserved yeast protein that likely serves an important function in chromatin structure and its relation to transcription. The experimental approaches emphasize the use of purified proteins and biochemical experiments to deduce molecular mechanisms. Genetic approaches in yeast are also utilized to test models developed from the biochemical studies and as exploratory tools. Finally DNA chip and phage display technologies are also utilized in specific cases. Given the high degree of conservation of gene regulatory proteins and general transcription factors among all eucaryotes, it seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply in other settings. A basic understanding of the molecular events underlying cell specialization provides not only a framework for understanding how the process can fail, but also provides the substrates and knowledge to design therapeutic strategies based on intervention.